Field of the Invention
The present invention relates to a method for size-dependent fractionation and determination of molecular mass (M) distribution of hyaluronan (HA) in biological samples.
Description of the Related Art
Hyaluronan (HA) is a polymeric extracellular glycosaminoglycan composed of repeating units of disaccharides of β-D-glucuronic acid and N-acetyl-β-D-glucosamine. HA is nearly ubiquitous in vertebrate solid and liquid tissues. It is a key macromolecular component of eye vitreous, articular joint synovial fluid, cartilage, and skin. HA is normally synthesized as a high molecular mass (M) polymer, generally ranging from about 1000-8000 kDa. HA synthase enzymes are integral membrane proteins of the cell surface, which synthesize HA as a pure polysaccharide polymer and extrude it into the extracellular matrix. HA chains can be released from the cell surface, or can be maintained as part of a pericellular layer, via noncovalent attachment to cell surface receptor proteins such as CD44. HA serves to organize proteoglycans of the pericellular and extracellular matrices via noncovalent binding interactions with their protein cores. It also controls the equilibrium partition and translational diffusion of other macromolecules near the cell surface via macromolecular crowding, and further maintains hydration of the pericellular matrix via osmotic pressure effects. In all of these properties, high M of HA is a critical requirement.
Assay of HA concentration in biological fluids such as blood serum has been shown to be useful in diagnosis of liver disease, because HA is normally rapidly removed from the blood by healthy liver. HA concentration in synovial fluid is reduced as fluid volume increases in osteoarthritis and rheumatoid arthritis, despite increased synthesis. The HA content of many solid tissues has been shown to increase in the presence of inflammation. Many disease states are accompanied by inflammation, and also have elevated HA levels. For example, lung carcinoma has greatly elevated levels of HA relative to normal lung tissue.
Reduction in the average M of HA in biological tissues and fluids is associated with inflammatory processes. Increased expression or activity of endogenous hyaluronidases can reduce the chain length of HA, and has been documented in various disease states. In addition, reactive oxygen and nitrogen species (ROS/RNS) are generated in inflammatory processes, and these can degrade HA by chemical means. Thus, low M HA can be generated by degradative processes, including those dependent on reactive oxygen and nitrogen species (ROS/RNS) and also by action of hyaluronidase enzymes. There are many different possible low M HA species, and the biological activity is believed to be dependent on the average M and polydispersity in M.
There is usually compensatory increase in HA synthesis, but the relative rates of HA synthesis and HA fragmentation determine the overall M distribution. When degradation exceeds synthesis, low M HA might exist in significant amounts.
Low M HA is a cell-signaling biomolecular species in biological tissues and fluids, believed to be a natural danger signal. Low M HA can be a natural stimulant for host defense against insults such as microbial attack. Response to low M HA is tissue-specific and dependent on the physiological state. Low M HA can regulate cell activity by interacting with receptor proteins, which causes signaling of a number of responses in an M-dependent manner, such as its signaling through cell surface receptors like CD44 and TLR2/4, as well as soluble HA receptors such as RHAMM. Clustering of cell surface CD44 when bound to polymeric high M HA is eliminated when bound to short HA fragments. This can lead to induction of cytokine and chemokine gene expression in macrophages, or cell death in activated T cells. The receptors CD44 and RHAMM bind low M HA in a manner leading to specific signaling processes. These include stimulation of NFκB signaling pathways and expression of pro-inflammatory cytokines and chemokines. Low M HA is believed to act as an endogenous danger signal, activating Toll-like receptors TLR2 and TLR4, and inducing changes in gene expression for mediators of host defense against microbes, mediators of inflammation response, and proteins connected with cell migration. HA signaling via TLR2/4 is speculated to occur by direct binding but this has not yet been proven.
The probable association of inflammation with the presence of low M HA indicates a need for improved and highly sensitive methods to accurately analyze the full M distribution of HA in biological tissues and fluids, including the fraction of low M.
The average M and distribution of M for HA present in biological sources has been studied primarily for fluid tissues such as synovial fluid, vitreous, serum, and lymph (Laurent and Granath, 1983; Dahl et al., 1985, 1986; Tengblad et al., 1986; Lee and Cowman, 1994; Armstrong and Bell, 2002). To date, the content of very low M HA (less than about 100 kDa) has been unidentified. Since low M HA is difficult to purify, specific detection of the separated HA is necessary. Because the low M HA is a signaling species, its content is also expected to be low and will require highly sensitive detection. Sandwich assays have proven incapable of detecting HA with M less than about 20 kDa, and have a severe M dependence of detection for HA between about 20 and 150 kDa (Yuan et al., 2013). Similarly, HA detection after blotting to membrane surfaces (used for the electrophoretic methods) has proven incapable of properly detecting HA with M less than about 150 kDa.
Most current methods for determination of the M distribution of HA from tissues and biological fluids have been optimized for high M HA (greater than about 200 kDa). Commonly employed methods are size exclusion chromatography with multiangle laser light scattering (SEC-MALLS), and agarose or polyacrylamide gel electrophoresis (Min et al., 1986; Kvam et al., 1993; Lee et al., 1994; Adam et al., 2001; Baggenstoss et al., 2006; Cowman et al., 2011; and Bhilocha et al., 2011). Detection of very low M HA by light scattering is inherently insensitive, and the SEC-MALLS method requires a highly purified HA sample. Gel electrophoresis can analyze samples on the microgram scale, and can tolerate some impurities in the sample, but nonspecific staining by those impurities can interfere with size distribution analysis of the HA. Blotting of gels to positively charged nylon and detection of HA using a labeled specific binding protein (Lee et al., 1994) works only for HA with M greater than about 100 kDa, as a result of strong surface binding (Yuan et al., 2013). Most alternative methods have similar limitations. Capillary electrophoresis (CE) (Hayase et al., 1997) is limited to pure HA samples. MALDI-TOF mass spectrometry (Mahoney et al., 2001 and Volpi et al., 2007) has high sensitivity, but requires a pure sample and HA with M larger than about 10 kDa becomes difficult to analyze (Yeung et al., 1999). The most promising method to date for complete size distribution analysis of HA isolated from biological samples is size exclusion chromatography-enzyme linked sorbent assay (SEC-ELSA) (Laurent et al., 1983; Tengblad et al., 1986; and Sasaki et al., 2011), because it is both sensitive and specific. However, SEC-ELSA has never been applied to the analysis of HA with M lower than about 100 kDa. A new method that has extremely high sensitivity and works best for low M HA is GEMMA (gas-phase electrophoretic mobility molecular analysis), but its accuracy has not yet been established for impure and polydisperse HA samples (Malm et al., 2012).
Anion exchange chromatography has not previously been applied to the determination of the M distribution for HA as isolated from biological sources, where there is a broad size distribution. Anion exchange chromatography with gradient elution using salt solutions of increasing ionic strength has been used previously only to separate short oligosaccharide fragments of HA by degree of polymerization and thus total charge (since there is one negative charge per disaccharide repeat unit). Success has been limited to fragments containing from 1 to about 20 disaccharides (0.4-8 kDa) (Weissmann, Meyer et al. 1954, Nebinger 1985, Holmbeck and Lerner 1993, Mahoney et al. 2001, Tawada et al. 2002). Short fragments of sulfated glycosaminoglycans, their desulfated products, or hybrid oligosaccharides created by transglycosylation have also been separated by anion exchange chromatography. Fragments containing 1 to approximately 20 disaccharides (ca. 10 kDa) were separated by size using an elution gradient of increasing ionic strength (Hoffman et al. 1956, Yamashina et al. 1963, Inoue and Nagasawa 1981, Lauder et al. 2000). No glycosaminoglycans have previously been fractionated by size/degree of polymerization/total charge for sizes above about 8-10 kDa. It has generally been expected that fragments larger than about 10 kDa would not be fractionated on the basis of size using ion exchange methods, due to small differences in total charge between long chains.
DNA and RNA oligonucleotides have been separated according to degree of polymerization over a much larger size range (Kato et al. 1983, Kato et al. 1988, Kato et al. 1989, Strege and Lagu 1991, Baba et al. 1993). DNA restriction fragments containing up to about 600 base pairs (ca. 390 kDa) could be separated into distinct peaks if fragment sizes differed by at least 5-10%. Further size-dependence of separation with low resolution could be achieved up to about 23,000 base pairs (ca. 15 MDa). The separation of high M DNA fragments used nonporous anion exchange media, because the large hydrodynamic volume of DNA molecules in solution makes entry into porous media difficult. Porous media may overlay a gel filtration separation mode on the ion exchange mode.
There is currently no facile method that has been shown to provide fractionation and specific quantification of HA over a broad M range including both low and high M.
Human milk provides newborns with a critical first line of natural defense against harmful infectious agents in addition to providing essential nutrition and factors that promote growth as well as organ and immune system development. Multiple components of milk, including carbohydrates, proteins, and fatty acids, work in concert to achieve protection against intestinal pathogens and formation of a beneficial microbiota that is essential to the future health of the baby (Newburg et al., 2005 and Ballard et al., 2013). Human milk oligosaccharides (HMOs) have been appreciated as beneficial glycans that promote healthy commensal bacteria and pathogen protection within the infant gut for over sixty years (Bode 2012). Similarly milk glycosaminoglycans have been shown to play a protective role, by inhibiting HIV infection (Newburg et al., 1995). Coppa et al., 2011 recently reported that hyaluronan (HA), a nonsulfated glycosaminoglycan extracellular matrix component produced by all vertebrates, is a natural component of human milk.
There are several challenges associated with the characterization of HA from human milk. Milk is a highly complex fluid, and standard HA isolation procedures are insufficient for removal of all contaminants. Even proteinase K cannot digest all of the protein components of milk. Initial efforts to analyze the molecular mass distribution of isolated milk HA by gel electrophoresis were unsuccessful, due to substantial staining interference by known (e.g., chondroitin or sulfated glycosaminoglycans) and unknown contaminants. The concentration of HA in milk is also very low, so that methods of high sensitivity are required for analysis of the isolated HA.
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